CN110922457B - Plant immune induced resistance protein FgPII1 secreted by fusarium graminearum and application thereof - Google Patents

Abstract

The invention discloses a plant immune induction protein FgPII1 secreted by fusarium graminearum and application thereof, wherein the protein can activate plant immune response and can be used as a plant immune induction factor to be applied to prevention and control of plant diseases. The amino acid sequence of the protein is shown as SEQ ID NO. 2. The high-concentration protein can be obtained by utilizing the over-expression of the engineering bacteria, and can be used for activating a tobacco immune system and improving the disease resistance of soybeans, and the related plant disease is soybean root rot. The plant immunity induced protein not only can obviously activate the plant immunity and improve the disease resistance of the plant, but also has quick response and low concentration. FgPII1 provides a new way for improving plant disease resistance and preventing and treating plant diseases.

Description

Plant immune induced resistance protein FgPII1 secreted by fusarium graminearum and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a plant immunity induced protein secreted by fusarium graminearum and application thereof.

Background

Fusarium graminearum (Fusarium graminearum) is a very devastating pathogenic fungus, and can infect various plants such as wheat, barley, soybean and the like, so that diseases such as wheat scab, soybean root rot and the like occur, and huge loss is caused to agricultural production (O' Donnell et al, 2004). Because of the lack of production of disease-resistant varieties against fusarium graminearum, chemical control is mainly relied on for plant diseases caused by the pathogen. However, the use of a large amount of chemical pesticides brings a series of problems of increased agricultural production cost, phytotoxicity, overproof pesticide residues, pathogen resistance, environmental pollution and the like, which not only seriously restricts the sustainable development of agriculture, but also endangers human health.

With the progress of technology and the improvement of research level, scientists gradually analyzed the relevant mechanisms of plant immunity and found that plants have a complete immune system (Jones and Dangl, 2006). During the process of invading plants by pathogenic bacteria, Pattern Recognition Receptors (PRRs) on the surfaces of plant cells can recognize extracellular factors secreted by the pathogenic bacteria and activate basic immunity of the plants. These extracellular factors capable of activating plant immunity are generally referred to as plant immune elicitors (Plant Immunity Inducer, PII). Plant immune elicitors are widely found in many microorganisms and play an important role in adaptation and survival of microorganisms. Plant immune elicitors are generally a very conserved group of substances, mainly consisting of polypeptide, protein, oligosaccharide, lipid, etc. types (boutroned zip., 2017). At present, with the proposal and development of the concept of green pesticide, the development of efficient and environment-friendly biopesticide is the trend of future development of pesticide, the principle of plant immunity for preventing and treating crop diseases and insect pests becomes an important development direction in the field of plant protection, and the identification of novel plant immunity inducers secreted by pathogenic bacteriaAnti-protein has also become the focus of attention of researchers at home and abroad.

A large body of literature indicates that it is possible to induce a plant immune response early in fusarium infestation in plants (Rep et al, 2004; Houterman et al, 2008; Shcherbakova et al, 2016; Thynne et al, 2017). Therefore, the identification of the novel plant immune resistance-inducing protein secreted by the fusarium graminearum not only can reveal the interaction mechanism of the fusarium graminearum and plants, but also can provide effective protein resources for developing protein biological pesticides for activating plant immunity.

Reference to the literature

Boutrot F and Zipfel C.Function,discovery,and exploitation of plant pattern recognitionreceptors for broad-spectrumdisease resistance.Annu.Rev.Phytopathol.2017.55:257–86.

Houterman PM,Cornelissen BJC and Rep M.Suppression of plant resistance gene-based immunity by a fungal effector.Plos Pathogens,2008,4(5):e1000061.

Jones JD and Dangl JL.The plant immune system.Nature,2006,444(7117):323-329.

O’Donnell K,Ward TJ,Geiser DM,et al.Genealogical concordance between the mating type locus and seven other nuclear genes supports formal recognition of nine phylogenetically distinct species within the Fusarium graminearum clade.Fungal Genet.Biol.,2004,41:600-623.

Rep M,Van Der Does H C,Meijer M,et al.A small,cysteine-rich protein secreted by Fusarium oxysporum during colonization of xylem vessels is required for I-3-mediated resistance in tomato.MolecμLar Microbiology,2004,53(5):1373-1383.

Shcherbakova LA,Odintsova TI,Stakheev AA,et al.Identification of a novel small cysteine-rich protein in the fraction from the biocontrol Fusarium oxysporum strain CS-20that mitigates Fusarium wilt symptoms and triggers defense responses in tomato.Frontiers in Plant Science,2016,6.

Thynne E,Saur IML,Simbaqueba J,et al.Fungal phytopathogens encode functional homologues of plant rapid alkalinization factor(RALF)peptides.MolecμLar Plant Pathology,2017,18(6):811-824.

Disclosure of Invention

One of the purposes of the invention is to provide a plant immunity inducing protein FgPII1 secreted by fusarium graminearum.

The second purpose of the invention is to provide a gene sequence for encoding plant immune induced resistance protein FgPII1 secreted by fusarium graminearum.

The invention also aims to provide a recombinant expression vector containing a plant immunity inducing protein FgPII1 encoding gene.

The fourth purpose of the invention is to provide a plant immunity inducing protein FgPII1 secreted by fusarium graminearum and application of the gene thereof.

The purpose of the invention can be realized by the following technical scheme:

a plant immune induction protein FgPII1 secreted by fusarium graminearum is (a) or (b) as follows:

(a) is represented by SEQ ID NO:2, and 2, or a pharmaceutically acceptable salt thereof;

(b) and (3) mixing the amino acid sequence shown in SEQ ID NO:2 and the amino acid residue sequence is subjected to substitution and/or deletion and/or addition of one or more amino acid residues, and the amino acid residue sequence is related to plant immunity induction and is represented by SEQ ID NO:2 derived protein.

A gene encoding the plant immunity-inducing protein FgPII 1; the gene is (1) or (2) as follows:

(1) a nucleotide sequence shown as SEQ ID NO. 1;

(2) a nucleotide sequence having at least 70% homology with SEQ ID NO. 1; preferably a nucleotide sequence having at least 80% or more homology with SEQ ID NO. 1; further preferably a nucleotide sequence having at least 85% homology with SEQ ID NO. 1; more preferably a nucleotide sequence having at least 90% or more homology with SEQ ID NO. 1; most preferred is a nucleotide sequence having at least 95% or more homology with SEQ ID NO. 1.

Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing the coding gene.

The recombinant vector is a prokaryotic expression vector obtained by inserting an encoding gene of FgPII1 into pGEX-4T-2. The prokaryotic expression vector can be used for the expression of Escherichia coli Rossetta (DE 3). The obtained fusion expression protein (GST-FgPII1) with the molecular weight of about 41KD can induce plants such as tobacco, soybean and the like to generate resistance reaction, improve the plant immunity and reduce the harm of soybean root rot caused by fusarium, pythium ultimum and phytophthora sojae.

By using the FgPII1 amino acid sequence, a nucleic acid sequence which is codon-optimized and is favorable for expression in escherichia coli can be designed and artificially synthesized.

The plant immunity inducing protein FgPII1, the coding gene or the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacterium are applied to inducing plant defense reaction and improving plant disease resistance.

The disease resistance is to plant diseases caused by fusarium, pythium ultimum and phytophthora sojae. The plant is tobacco, soybean, etc.

A method for preventing and controlling plant diseases, wherein the plant is treated by the plant immunity inducing protein FgPII1 to prevent and control the plant diseases caused by fusarium, pythium ultimum and phytophthora sojae.

The plant immunity inducing protein FgPII1 provided by the invention is applied to inducing plant defense reaction and improving plant disease resistance. The disease resistance is against plant diseases caused by fusarium, pythium ultimum and phytophthora sojae, such as soybean root rot. When used to induce plant resistance, reducing the soybean root rot hazard, the concentration of FgPII1 was 1 nM.

The plant immunity induced protein can be protein obtained by prokaryotic expression of a genetic engineering technology, and can also be protein obtained by purifying fusarium graminearum culture solution. With the same effect.

The invention has the beneficial effects that:

the plant immunity induced protein can obviously improve the disease resistance of plants, and has low use concentration, quick response and long action time. FgPII1 provides a new way for improving the resistance of plants by using the immune system of the plants, thereby having wide application prospect in agricultural production.

Drawings

FIG. 1 is a graph of the detection of allergic necrosis in tobacco leaves induced after expression of the plant immune-inducing protein FgPII1 on tobacco leaves;

FIG. 2 is a Western blot assay of FgPII1 for normal expression on tobacco leaves using anti-HA;

FIG. 3 is a Western blot detection diagram of plant immune induced resistance protein FgPII1 obtained after expression and purification by a prokaryotic expression system, wherein the antibody is anti-GST;

FIG. 4 shows that after the plant immunity inducing protein FgPII1 is injected to treat tobacco leaves, the expression of the genes related to tobacco disease resistance is induced;

FIG. 5 is a graph showing the onset of phytophthora sojae after treatment of soybean hypocotyls with the plant immunity-inducing protein FgPII 1;

FIG. 6 is a statistical plot of biomass of plants after immunization against protein FgPII1 treatment of soybean hypocotyls and inoculation with Phytophthora sojae;

FIG. 7 is a graph showing the onset of Fusarium graminearum after treatment of soybean hypocotyls with the plant immunity-inducing protein FgPII 1;

FIG. 8 is a statistical plot of biomass of a plant immunity inducing protein FgPII1 after treatment of soybean hypocotyls and inoculation with Fusarium graminearum;

FIG. 9 is a graph showing the onset of Pythium ultimum inoculation after soybean hypocotyls are treated with the plant immunity-inducing protein FgPII 1;

FIG. 10 is a statistical plot of biomass after treatment of soybean hypocotyls with the plant immunity inducing protein FgPII1 and inoculation of Pythium ultimum.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The primer related to the embodiment of the invention is synthesized by Nanjing Kingsrei Biotechnology GmbH.

Example 1 isolation and characterization of plant immune response-inducing protein FgPII1

Inoculating fusarium graminearum to a PDA (potato peeling is taken 200g, cut into small blocks, added with ultrapure water and boiled for 30min, four layers of gauze are used for filtering to remove potato blocks, 20g of glucose and 15g of agar powder are added into filtrate, the filtrate is replenished to 1000mL with ultrapure water, split charging is carried out after dissolution, and high-pressure steam sterilization is carried out at 121 ℃ for 30min) flat plate, culturing for 7 days at 25 ℃, selecting the edges of bacterial colonies, inoculating the bacterial colonies into 200mL of 500mL triangular flasks filled with PDB (PDA culture medium with the same formula and without agar powder), and culturing for 5 days in a shaking table at 25 ℃ and 180r/min to obtain the fusarium graminearum culture solution. The culture filtrate was centrifuged at 8000rpm at 4 ℃ for 30min, and the supernatant was collected and further filtered through a 0.22. mu.M filter (Whatman) to remove impurities. And (3) collecting 50mL of culture solution to obtain filtrate, and sending the filtrate to Shenzhen Hua DageneCo for proteomic analysis. And (3) comparing data obtained by proteomics analysis with a fusarium graminearum protein database based on the fact that the fusarium graminearum genome is sequenced, and determining 7 candidate proteins secreted by fusarium graminearum in the culture filtrate.

Example 2 cloning and transient expression of the Gene encoding the plant immune response-inducing protein FgPII1

(1) Total RNA extraction: taking fusarium graminearum hyphae cultured in liquid as a material, extracting total RNA by adopting an Omega RNA extraction kit according to the instructions, and detecting the RNA content and quality by using a spectrophotometer.

(2) Reverse transcription to generate the first strand: mu.g of RNA was used as a template, and cDNA synthesis was carried out according to the instructions of the kit for PrimeScript reverse transcriptase of Takara, Inc., and the volume was adjusted to 20. mu.L. Appropriate amounts of the reverse transcription products were taken for subsequent gene cloning PCR.

(3) And (3) performing PCR by using the first strand of the cDNA as an RT-PCR template by a conventional method to amplify the full length of the FgPII1 coding gene:

PCR primer amplification sequence:

an upstream primer: SEQ ID NO.3

(5’-CAGCTAGCATCGATTCCC ATGCTCTTCTTCAAGTCTAT-3’)

A downstream primer: SEQ ID NO.4

(5’-AATCTCTAGAGGATCCCC GCTAGTGCCAGTGCAGCCA-3’)，

A50. mu.L reaction system in which 5 XBuffer 10. mu.L, 2.5mM dNTPs 4. mu.L, Takara PrimerSTARTaq enzyme 0.5. mu.L, template cDNA 1. mu.L, water to 50. mu.L; the PCR amplification program comprises pre-denaturation at 98 ℃ for 3min, denaturation at 98 ℃ for 15 seconds, annealing at 58 ℃ for 15 seconds, extension at 72 ℃ for 30 seconds, circulation for 35 times and extension at 72 ℃ for 10 min; and detecting whether the obtained band is a target band by agarose gel electrophoresis and Ethidium Bromide (EB) staining, and further cutting the gel to recover a PCR product of the FgPII1 encoding gene. The electrophoretic bands were recovered using the Agarose Gel DNAPurification Kit (TaKaRa) Kit. The obtained PCR product of the gene encoding FgPII1 was ligated to SmaI digested pGR107::3HA vector according to the Kit instructions of Cloneexpress II One Step Cloning Kit (Vazyme) to obtain pGR107:: FgPII1-3HA plasmid, further transformed into Escherichia coli competent cell JM109, spread on LB (containing 50. mu.g/mL) plate, cultured at 37 ℃ for 16h, subjected to colony PCR verification, selected positive clones, extracted according to the plasmid extraction Kit procedure (Takara), sent to the company for sequencing (Shanghai Biotechnology), and the sequence was as SEQ ID NO. 1. The plasmid with correct sequencing is transformed into agrobacterium GV3101 by electric shock, coated on LB (containing kanamycin 50 mug/mL, rifampicin 50 mug/mL) plate, cultured at 28 ℃, and colony PCR verified after 48h, and the correct clone is picked for subsequent experiments.

(4) Agrobacterium culture

Single colonies of Agrobacterium GV3101 transfected with pGR107:: FgPII1-3HA vector and pGR107:: GFP-3HA vector were picked from plates and inoculated into 2mL LB liquid medium (containing kanamycin 50. mu.g/mL, rifampicin 50. mu.g/mL) at 28 ℃ on a constant temperature shaker at 200rpm overnight to OD600 of 2.0. The overnight cultured GV3101 Agrobacterium solution was centrifuged at 5000g for 3min to collect the cells. Buffer (composition: 10mM 2- [ N-morpholino)]ethanesulfonic acid,10mM MgCl₂200 μ M acetosyringone pH 5.6) suspension of the bacterial solution and centrifugation to collect the cells. After repeated washing 2 times, the bacterial solution was diluted with buffer to a final concentration of 0.5 each.

(5) Tobacco leaf sheet transient expression FgPII1 encoding gene

The Agrobacterium diluted to a certain concentration was injected into tobacco leaves with 1mL syringe without needle, and the tobacco was cultured in a greenhouse (21-23 ℃ C., 14h light/10 h dark) after injection.

(6) FgPII1 protein accumulation detection

Collecting tobacco leaves two days after injection for protein expression amount detection. The collected tobacco leaves were frozen with liquid nitrogen, ground, added with a protein extract (consisting of 150mM NaCl,50mM Tris-HCl pH 7.5, 1.0% (v/v) NP-40, 1.0% (v/v) protease inhibitor cocktail), and mixed well on ice for 30 min. 18000g, centrifuging, collecting supernatant 80. mu.L, adding 20. mu.L of 5 times protein loading buffer solution, mixing, and boiling in water bath for 5 min. A10. mu.L sample was run on SDS-PAGE gels for 1.5h at 120V. After the reaction, the protein sample was transferred to PVDF membrane, and the membrane was sealed by incubating with 5% PBST milk. The membrane was washed with PBST three times after 2h incubation with 1:5000 dilution of HA primary antibody (Abmart), followed by 30min incubation with 1:10000 dilution of murine antibody (LI-COR, irdye 800, 926-.

As a result: three days after transient expression of FgPII1 on tobacco leaves, a clear allergic necrotic reaction appeared on tobacco leaves (see FIG. 1). Western detection confirmed that FgPII1 was normally expressed (see FIG. 2).

Example 3 prokaryotic expression and purification of plant immune response-inducing protein FgPII1

(1) Construction of prokaryotic expression vector

Designing a specific primer of the coding gene of the plant immune induced resistance protein FgPII1,

an upstream primer: SEQ ID NO.5

(5’-TCCCCAGGAATTCCC ATGAGCCCCATCCTCGAGACC-3’)

A downstream primer: SEQ ID NO.6

(5’-CGCTCGAGTCGACCCGCTAGTGCCAGTGCAGCCA-3’)，

50 μ L reaction: wherein 5 XBuffer 10. mu.L, 2.5mM dNTPs 4. mu.L, Takara PrimerSTARTaq enzyme 0.5. mu.L, template cDNA 1. mu.L, water to 50. mu.L; the PCR amplification program comprises pre-denaturation at 98 ℃ for 3min, denaturation at 98 ℃ for 15 seconds, annealing at 58 ℃ for 15 seconds, extension at 72 ℃ for 40 seconds, circulation for 35 times, and final extension at 72 ℃ for 10 min; the agarose gel was subjected to electrophoretic separation, photographed by Ethidium Bromide (EB) staining, the results were recorded, and the PCR product of the gene encoding FgPII1 was recovered by cutting the gel. The electrophoretic band was recovered with an Agarose Gel DNAPuration Kit (TaKaRa). PCR products of FgPII1 encoding genes recovered by gel cutting are connected to a SmaI enzyme-cut pGEX-4T-2 vector according to the instruction operation of Cloneexpress II One Step Cloning Kit (Vazyme) to obtain pGEX-4T-2-FgPII1 plasmid, Escherichia coli competent cell JM109 is transformed, LB (containing 50 ug/mL) plates are coated, after 16h of culture at 37 ℃, colony PCR verification is carried out, three positive clones are picked up to extract the plasmid according to the operation of a plasmid extraction Kit (Takara), and the plasmid is sent to Nanjing Kingsry company for sequencing, and the sequence is shown as SEQ ID NO. 1. Sequencing of the correct plasmid Heat shock transformed E.coli Rosseta (DE3) competent cells were plated on LB (containing 50. mu.g/mL) plates, incubated at 37 ℃ for 16h, followed by colony PCR verification and positive clones were picked for subsequent experiments.

(2) Protein induced expression

The correct expression strains were activated overnight while using the strain containing the empty plasmid pGEX-4T-2 as a control. Adding 1mL of overnight culture bacterial liquid into 100mL of LB liquid culture medium (2% inoculum size) containing 50 mu g/mL of ampicillin, carrying out shake culture at 37 ℃ and 200r/min for 2-3 h until OD600 is 0.6-0.8, adding inducer IPTG (final concentration is 1mM), and carrying out shake culture at 20 ℃ and 220r/min for 16h to induce and express target protein. And (3) high-speed centrifugation, collecting thalli, adding a buffer solution into the thalli, crushing thalli by using the obtained thalli suspension, high-speed centrifugation at 4 ℃, and collecting supernatant to obtain the expression protein solution. And (3) adding 5 mu L of 5 xSDS protein loading buffer into 20 mu L of supernatant, heating in a boiling water bath for 10min, centrifuging at 13000r/min for 10min, taking the supernatant, carrying out SDS-PAGE detection, and carrying out Coomassie blue staining.

As a result: through SDS-PAGE detection, a fusion expression protein (GST-FgPII1) containing GST with the molecular weight of about 41kD is obtained.

(3) Purification of prokaryotic expression proteins

Purification of the prokaryotic expression protein was performed using an AKTA Explorer 100 protein purifier. Selecting a GST tag protein purification column for affinity chromatography, and balancing the affinity chromatography column by using a cleaning buffer solution; and (3) sampling the protein solution obtained in the step (2), carrying out elution by using an elution buffer solution when the flow rate is 1mL/min until the base line is stable, collecting an elution peak, desalting the components of the elution peak by using an ultrafiltration tube, and then carrying out SDS-PAGE electrophoresis to detect the purity of the protein.

As a result: the purified prokaryotic expression protein (GST-FgPII1) was obtained in the size of about 41kD as shown in FIG. 3.

Example 4 defence response on tobacco by the plant immune response-inducing protein FgPII1

(1) FgPII1 induces the transcription level of resistance related gene to be obviously increased

The concentration of FgPII1 protein was adjusted to 1. mu.M after the concentration measurement. Selecting the middle leaf of the tobacco growing vigorously in the 5-6 stages, and injecting FgPII1 protein into different leaves from the back of the leaf by using a 1mL syringe without a needle. Meanwhile, pGEX-4T-2 (expression empty vector protein) with the same concentration is used as a control, and after 6 hours of injection, samples are collected for detecting the transcription level of the resistance related gene. Total RNA extraction was performed using an Omega RNA extraction kit according to the instructions, and the RNA content and quality were measured using a spectrophotometer.

First strand generation by reverse transcription 0.7. mu.g of RNA was used as a template, and cDNA synthesis was carried out according to the instructions of the kit for PrimeScript reverse transcriptase from Takara, to a volume of 20. mu.L. The reverse transcription product was diluted 10-fold with water for real-time quantitative PCR reaction to detect gene silencing efficiency.

The primers used in the real-time fluorescent quantitative PCR reaction were as follows:

NbCYP71D20 upstream primer: SEQ ID NO.7

5’-CCGCACCATGTCCTTAGAG-3’

NbCYP71D20 downstream primer: SEQ ID NO.8

5’-CTTGCCCCTTGAGTACTTGC-3’

NbAcre31 upstream primer: SEQ ID NO.9

5’-AATTCGGCCATCGTGATCTTGGTC-3’

NbAcre31 downstream primer: SEQ ID NO.10

5’-GAGAAACTGGGATTGCCTGAAGGA-3’

NbWRKY7 upstream primer: SEQ ID NO.11

5’-CACAAGGGTACAAACAACACAG-3’

NbWRKY7 downstream primer: SEQ ID NO.12

5’-GGTTGCATTTGGTTCATGTAAG-3’

NbEF1a upstream primer: SEQ ID NO.13

5’-TGGTGTCCTCAAGCCTGGTA-3’

NbEF1a downstream primer: SEQ ID NO.14

5’-ACGCTTGAGATCCTTAACCGC-3’

The PCR reaction system contained 5. mu.L of cDNA, 10. mu.L of SYBR Premix Ex Taq II (Tli RNase H Plus), 0.4. mu.L of the front and back primers, 0.4. mu.L of ROX Reference Dye II, and 13.8. mu.L of water. Reaction procedure: i95 ℃ for 30 seconds, II 95 ℃ for 5 seconds, 60 ℃ for 34 seconds, step II for 40 cycles. The dissolution curve analysis program was: 95 ℃ for 15 seconds, 60 ℃ for 1 minute, 95 ℃ for 15 seconds. The data analysis adopts a 2-delta CT method, and the detection result is shown in figure 6. The references Livak, K.J., and Schmittgen, T.D, (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2- Δ CT method, methods 25, 402-.

As a result: the fluorescent quantitative PCR result shows that the expression level of the induced tobacco disease-resistant related gene is remarkably increased 6h after the tobacco leaves are treated by 1 mu M FgPII1 protein (as shown in figure 4).

Example 5 plant immune response-inducing protein FgPII1 enhances disease resistance in Soybean

(1) FgPII1 enhances resistance of soybeans to phytophthora sojae

Diluting the purified FgPII1 solution to 1nM, soaking soybean etiolation seedlings growing for 5 days into 1nM FgPII1 solution, shading treatment in a culture room at 25 ℃ for 12h, taking out the etiolation seedlings, inoculating phytophthora sojae, shading and moisturizing in the culture room at 25 ℃ for 2 days, and then observing and taking pictures of disease symptoms (figure 5). Repeat 3 times for each 10 strains treated. And sampling and utilizing fluorescent quantitative PCR to carry out phytophthora sojae infection biomass measurement, wherein the method is the same as the example 4), and the PsActin gene is selected as the phytophthora sojae internal reference gene, and the GmCYP2 gene is the soybean internal reference gene. The quantitative primer sequences were as follows:

GmCYP2 upstream primer: SEQ ID NO.15

5’-CGGGACCAGTGTGCTTCTTCA-3’

Downstream primer of GmCYP 2: SEQ ID NO.16

5’-CCCCTCCACTACAAAGGCTCG-3’

PsActin upstream primer: SEQ ID NO.17

5’-ACTGCACCTTCCAGACCATC-3’

PsActin downstream primer: SEQ ID NO.18

5’-CCACCACCTTGATCTTCATG-3’

As a result: compared with the empty carrier protein control expressed by pGEX-4T-2, the soybean etiolation seedlings treated by FgPII1 all showed significant reduction of lesion length after inoculation of phytophthora sojae, and the biomass of phytophthora sojae infection was significantly reduced (FIG. 6).

(2) FgPII1 enhances resistance of soybeans to Fusarium graminearum

Diluting the purified FgPII1 solution to 1nM, soaking soybean etiolation seedlings growing for 5 days in 1nM FgPII1 solution, shading treatment in a culture room at 25 ℃ for 12h, taking out the etiolation seedlings, inoculating fusarium graminearum, shading and moisturizing in a culture room at 25 ℃ for 2 days, and observing and taking a picture of onset symptoms (figure 7). Repeat 3 times for each 10 strains treated. And the samples were taken and the amount of fusarium graminearum infecting organisms was determined by fluorescent quantitative PCR, in the same manner as in example (4). The FgCYP gene was selected as a reference gene for Fusarium graminearum. The quantitative primer sequences were as follows:

FgCYP upstream primer: SEQ ID NO.19

5’-GTCCAATCCACTCCATCCTC-3’

FgCYP downstream primers: SEQ ID NO.20

5’-CGGTCTTCTCGAGAGGTTCA-3’

As a result: compared to the empty carrier protein control expressed by pGEX-4T-2, soybean yellowing seedlings treated with FgPII1 all showed a significant reduction in lesion length and a significant reduction in biomass of Fusarium graminearum infection after inoculation with Fusarium oxysporum (FIG. 8).

(3) FgPII1 enhances resistance of soybeans to Pythium ultimum

Diluting the purified FgPII1 solution to 1nM, soaking soybean etiolation seedlings growing for 5 days in 1nM FgPII1 solution, shading treatment in a 25 ℃ culture room for 12h, taking out the etiolation seedlings, inoculating Pythium ultimum, shading and moisturizing in a 25 ℃ culture room for 2 days, and photographing for disease symptom observation (figure 9). Repeat 3 times for each 10 strains treated. And sampling and using fluorescent quantitative PCR to determine the Pythium ultimum infection biomass, the method is the same as the example (4). The primer sequence of the pythium ultimum internal reference gene quantification is as follows: pu upstream primer: SEQ ID NO.21

5’-CAACTGGAAAAGCAAGCGG-3’

Pu downstream primer: SEQ ID NO.22

5’-CCGAAGAACTGTGTCCGC-3’

As a result: compared to the empty carrier protein control expressed by pGEX-4T-2, soybean yellowing seedlings treated with FgPII1 all showed a significant reduction in lesion length and a significant reduction in biomass of Fusarium graminearum infection after inoculation with Fusarium oxysporum (FIG. 10).

Sequence listing

FgPII1 coding gene full-length nucleotide sequence: SEQ ID NO.1(DNA sequence) Fusarium graminearum (Fusarium graminearum) with sequence length 399

ATGCTCTTCTTCAAGTCTATCGTCTCTCTCGCAGCCCTCGTTGGCGTTGCTGTTGCCAGCCCCATCCTCGAGACCCGCCAGTCTGCCACTCGCTGCGGCAGCACCAGCTACACTGCTGCTCAGGTCAAGGCCGCTGCCAACGCCGCGTGCCAGTACTACCAGAATGATGATACCGCTGGTAGCTCGACTTACCCTCATCAATACAACAACCGAGAGGGCTTTGACTTTCCTGTGAACGGTCCTTACCAAGAGTTCCCCATCCGAACTAGCGGTGTTTACACCGGAGGCTCGCCCGGTGCCGACCGTGTTGTTATCAACACCAACTGTCAATTCGCCGGTGCTATCACCCATACCGGCGCTTCTGGAAACAACTTTGTTGGCTGCACTGGCACTAGCTAA

Full-length protein sequence of FgPII 1: SEQ ID NO.2 (amino acid sequence) Fusarium graminearum (Fusarium graminearum) sequence length 132

MLFFKSIVSLAALVGVAVASPILETRQSATRCGSTSYTAAQVKAAANAACQYYQNDDTAGSSTYPHQYNNREGFDFPVNGPYQEFPIRTSGVYTGGSPGADRVVINTNCQFAGAITHTGASGNNFVGCTGTS

SEQ ID NO.3(DNA Artificial sequence) sequence length 38

cagctagcatcgattcccatgctcttcttcaagtctat

SEQ ID NO.4(DNA Artificial sequence) sequence length 37

aatctctagaggatccccgctagtgccagtgcagcca

SEQ ID NO.5(DNA Artificial sequence) sequence length 36

tccccaggaattcccatgagccccatcctcgagacc

SEQ ID NO.6(DNA Artificial sequence) sequence length 34

cgctcgagtcgacccgctagtgccagtgcagcca

SEQ ID NO.7(DNA Artificial sequence) sequence length 19

ccgcaccatgtccttagag

SEQ ID NO.8(DNA Artificial sequence) sequence length 20

cttgccccttgagtacttgc

SEQ ID NO.9(DNA Artificial sequence) sequence length 24

aattcggccatcgtgatcttggtc

SEQ ID NO.10(DNA Artificial sequence) sequence length 24

gagaaactgggattgcctgaagga

SEQ ID NO.11(DNA Artificial sequence) sequence length 22

cacaagggtacaaacaacacag

SEQ ID NO.12(DNA Artificial sequence) sequence length 22

ggttgcatttggttcatgtaag

SEQ ID NO.13(DNA Artificial sequence) sequence length 20

tggtgtcctcaagcctggta

SEQ ID NO.14(DNA Artificial sequence) sequence length 21

acgcttgagatccttaaccgc

SEQ ID NO.15(DNA Artificial sequence) sequence length 21

cgggaccagtgtgcttcttca

SEQ ID NO.16(DNA Artificial sequence) sequence length 21

cccctccactacaaaggctcg

SEQ ID NO.17(DNA Artificial sequence) sequence length 20

actgcaccttccagaccatc

SEQ ID NO.18(DNA Artificial sequence) sequence length 20

Ccaccaccttgatcttcatg

SEQ ID NO.19(DNA Artificial sequence) sequence length 20

gtccaatccactccatcctc

SEQ ID NO.20(DNA Artificial sequence) sequence length 20

cggtcttctcgagaggttca

SEQ ID NO.21(DNA Artificial sequence) sequence length 20

caactggaaaagcaagcgg

SEQ ID NO.22(DNA Artificial sequence) sequence length 20

ccgaagaactgtgtccgc

Sequence listing

<110> Nanjing university of agriculture

<120> plant immune induced resistance protein FgPII1 secreted by fusarium graminearum and application thereof

<160> 22

<170> SIPOSequenceListing 1.0

<210> 1

<211> 399

<212> DNA

<213> Fusarium graminearum (Fusarium graminearum)

<400> 1

atgctcttct tcaagtctat cgtctctctc gcagccctcg ttggcgttgc tgttgccagc 60

cccatcctcg agacccgcca gtctgccact cgctgcggca gcaccagcta cactgctgct 120

caggtcaagg ccgctgccaa cgccgcgtgc cagtactacc agaatgatga taccgctggt 180

agctcgactt accctcatca atacaacaac cgagagggct ttgactttcc tgtgaacggt 240

ccttaccaag agttccccat ccgaactagc ggtgtttaca ccggaggctc gcccggtgcc 300

gaccgtgttg ttatcaacac caactgtcaa ttcgccggtg ctatcaccca taccggcgct 360

tctggaaaca actttgttgg ctgcactggc actagctaa 399

<210> 2

<211> 132

<212> PRT

<213> Fusarium graminearum (Fusarium graminearum)

<400> 2

Met Leu Phe Phe Lys Ser Ile Val Ser Leu Ala Ala Leu Val Gly Val

1 5 10 15

Ala Val Ala Ser Pro Ile Leu Glu Thr Arg Gln Ser Ala Thr Arg Cys

20 25 30

Gly Ser Thr Ser Tyr Thr Ala Ala Gln Val Lys Ala Ala Ala Asn Ala

35 40 45

Ala Cys Gln Tyr Tyr Gln Asn Asp Asp Thr Ala Gly Ser Ser Thr Tyr

50 55 60

Pro His Gln Tyr Asn Asn Arg Glu Gly Phe Asp Phe Pro Val Asn Gly

65 70 75 80

Pro Tyr Gln Glu Phe Pro Ile Arg Thr Ser Gly Val Tyr Thr Gly Gly

85 90 95

Ser Pro Gly Ala Asp Arg Val Val Ile Asn Thr Asn Cys Gln Phe Ala

100 105 110

Gly Ala Ile Thr His Thr Gly Ala Ser Gly Asn Asn Phe Val Gly Cys

115 120 125

Thr Gly Thr Ser

130

<210> 3

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cagctagcat cgattcccat gctcttcttc aagtctat 38

<210> 4

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aatctctaga ggatccccgc tagtgccagt gcagcca 37

<210> 5

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tccccaggaa ttcccatgag ccccatcctc gagacc 36

<210> 6

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cgctcgagtc gacccgctag tgccagtgca gcca 34

<210> 7

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ccgcaccatg tccttagag 19

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cttgcccctt gagtacttgc 20

<210> 9

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aattcggcca tcgtgatctt ggtc 24

<210> 10

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gagaaactgg gattgcctga agga 24

<210> 11

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cacaagggta caaacaacac ag 22

<210> 12

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ggttgcattt ggttcatgta ag 22

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tggtgtcctc aagcctggta 20

<210> 14

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

acgcttgaga tccttaaccg c 21

<210> 15

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cgggaccagt gtgcttcttc a 21

<210> 16

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

cccctccact acaaaggctc g 21

<210> 17

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

actgcacctt ccagaccatc 20

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ccaccacctt gatcttcatg 20

<210> 19

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gtccaatcca ctccatcctc 20

<210> 20

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

cggtcttctc gagaggttca 20

<210> 21

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

caactggaaa agcaagcgg 19

<210> 22

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

ccgaagaact gtgtccgc 18

Claims (6)

1. As shown in SEQ ID NO:2 in inducing plant defense reaction and improving plant disease resistance.

2. As shown in SEQ ID NO: 1 in the expression of.

3. Comprises the nucleotide sequence shown in SEQ ID NO: 1, and the application of a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium of the coding gene of the plant immunity inducing protein FgPII1 in inducing plant defense reaction and improving plant disease resistance.

4. The use according to claim 1, 2 or 3, wherein the disease resistance is against plant diseases caused by Fusarium, Pythium ultimum and Phytophthora sojae.

5. Use according to claim 1, 2 or 3, wherein the plant is tobacco or soybean.

6. A method for controlling plant diseases, characterized in that the plant diseases are controlled by the nucleic acid sequences shown in SEQ ID NO:2, and the plant immunity inducing protein FgPII1 is used for treating plants to prevent and control plant diseases caused by fusarium, pythium ultimum and phytophthora sojae.

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